Lecture 18

Question 1

What happens in cloning?

Answer 1

A sequence of DNA is inserted into a plasmid vector and this vector can then be put into a cell

Question 2

What is the aim of cloning?

Answer 2

To obtain isolated and purified copies of specific gene sequences

Question 3

What do cloned genes allow us to do?

Answer 3

• Determine the sequence of the gene (and protein)
• Obtain leads into the function of the gene
• Manipulate the gene
• > mutate, insert gene into cells of organism, make large amounts of protein

Question 4

What is a polylinker?

Answer 4

(A multiple cloning site)

-String of joined restriction enzyme sites

Question 5

What must a vector have?

Answer 5

1. An origin of replication
2. A selectable marker
3. Unique restriction enzyme cleavage sites

Question 6

What are the steps of Cloning?

Answer 6

1. RESTRICTION - plasmid vector and DNA fragment of interest must be digested with a restriction enzyme to produce sticky ends
2. LIGATION - DNA fragment of interest must be ligated into a plasmid vector
3. TRANSFORMATION - ligated plasmid vector must be transformed into a bacterial host
4. SELECTION - bacterial colonies carrying the plasmid with DNA fragment must be selected

Question 7

What will staggered REnzyme cuts give you?

Answer 7

Sticky ends

Question 8

What will blunt REnzyme cuts give you?

Answer 8

Blunt ends

Question 9

Can 2 different enzymes be used to make the cuts?

Answer 9

No, the same restriction enzyme must be used to cut the vector and in the insert DNA

Question 10

How is the DNA ligated?

Answer 10

Ligase enzyme covalently joins the DNA

Question 11

What are the possible results of mixing the restricted plasmid vector and DNA in bacterial cells?

Answer 11

1. Uncut/re-ligated only = grows BLUE
2. Un-ligated insert only = no growth
3. Cut vector = no growth
4. Correct with insert in vector = growth WHITE

Question 12

What is the function of Beta-lactamase?

Answer 12

Cleaves ampicillin (an antibiotic)

Question 13

How would you screen for antibiotic resistance?

Answer 13

• Plate ligation products onto Ampicillin to select bacteria with plasmids
• Products = uncut (no vector) and correct insert (vector)

Question 14

What is the alpha-peptide used for?

Answer 14

Used to make part of the beta-gal

-A-peptide stabilised B-gal and enables cleavage of X-gal (an analogue of lactose)

Question 15

What does cleavage of X-gal cause?

Answer 15

Blue product

Question 16

Describe the two possible events regarding B-gal and the alpha peptide

Answer 16

1. B-gal WITH a-peptide -> cleaves X-gal -> Blue product
   (no insert)
2. B-gal WITHOUT a-peptide -> cannot cleave X-gal -> White product
   (DNA insert in the Lac Z a-gene)

Question 17

Ampicillin selects bacteria with an BLANK plasmid

Answer 17

An intact

Question 18

X-gal selects plasmids that ….

Answer 18

Have an insert